U.S. patent number 6,042,874 [Application Number 09/051,031] was granted by the patent office on 2000-03-28 for process for processing organic specimens.
This patent grant is currently assigned to Milestone S.r.l.. Invention is credited to Werner Lautenschlager, Francesco Visinoni.
United States Patent |
6,042,874 |
Visinoni , et al. |
March 28, 2000 |
**Please see images for:
( Certificate of Correction ) ** |
Process for processing organic specimens
Abstract
This invention relates to the processing of organic tissues to
prepare them for further investigation such as by light microscopy,
electron microscopy, microwave staining, immunostaining,
decalcification, hybridisation etc. The present invention discloses
a process for processing organic tissue, which comprises the steps
of fixing an organic sample to be processed, dehydrating and
clearing the sample and subsequently infiltrating and embedding the
sample in a supporting medium to prepare it for further examination
wherein the steps of dehydrating and clearing are carried out
simultaneously by applying a mixture of a dehydration agent and an
essentially lipophilic agent simultaneously to the sample.
Inventors: |
Visinoni; Francesco (Bergamo,
IT), Lautenschlager; Werner (Leutkirch,
DE) |
Assignee: |
Milestone S.r.l. (Bergamo,
IT)
|
Family
ID: |
8223071 |
Appl.
No.: |
09/051,031 |
Filed: |
November 23, 1998 |
PCT
Filed: |
August 01, 1997 |
PCT No.: |
PCT/EP97/04207 |
371
Date: |
November 23, 1998 |
102(e)
Date: |
November 23, 1998 |
PCT
Pub. No.: |
WO98/05938 |
PCT
Pub. Date: |
February 12, 1998 |
Foreign Application Priority Data
|
|
|
|
|
Aug 2, 1996 [EP] |
|
|
96112571 |
|
Current U.S.
Class: |
427/2.11;
427/296; 427/4 |
Current CPC
Class: |
G01N
1/30 (20130101) |
Current International
Class: |
G01N
1/30 (20060101); A01N 001/00 (); B05D 001/34 () |
Field of
Search: |
;427/4,2,11,336,595,296,553 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
The Two-Step Vacuum-Microwave Method for Histoprocessing Mathilde
E. Boon; C.H. Wals-Paap; F.A. Visinoni; L.P. Kok European Journal
of Morphology, vol. 33, No. 4, 1995 Microwave Newsletter p. 349-358
XP 000614291 (no month)..
|
Primary Examiner: Dudash; Diana
Attorney, Agent or Firm: Lacasse & Associates Lacasse;
Randy W. Strickland; Wesley L.
Claims
We claim:
1. A process for processing tissue comprising the steps of
(i) fixing an organic sample to be processed,
(ii) dehydrating the sample with a dehydration agent,
(iii) clearing the sample with an essentially lipohilic agent,
and
(iv) impregnating the sample,
wherein said steps (ii) and (iii) are carried out simultaneously by
applying a mixture of said dehydration agent and said essentially
lipophilic agent simultaneously to the sample.
2. The process according to claim 1, wherein said sample comprises
organic tissue.
3. The process according to claim 2, wherein said organic tissue is
from a group comprising blood, nerve tissue and organ tissue.
4. The process according to claim 1, wherein the process further
comprises between said steps (ii/iii) and (iv) a step (v) of drying
the sample at reduced pressure.
5. The process according to claim 2, wherein the process further
comprises between said steps (ii/iii) and (iv) a step (v) of drying
the sample at reduced pressure.
6. The process according to claim 2, wherein the dehydration agent
is selected from the group consisting of C.sub.1-8 -alcohols,
C.sub.1-8 -ethers, ketones and phenols and anilines,
tetrahydrofuran, 2,2-dimethoxypropane and 2,2-diethoxypropane and
creosote or a mixture thereof.
7. The process according to claim 2, wherein the alcohol is
selected from the group consisting of methanol, ethanol, propanol,
isopropanol, n-butanol and t-butanol or a mixture thereof.
8. The process according to claim 2, wherein the ether is selected
from the group consisting of glycolethers, 2-ethoxyethanol, dioxane
and polyethylene glycol or a mixture thereof.
9. The process according to claim 2, wherein the ketone is
acetone.
10. The process according to claim 2, wherein the essentially
lipophilic agent is selected from the group consisting of
aliphatic, aromatic and chlorinated hydrocarbons, alkanes, esters
and terpenes or a mixture thereof.
11. The process according to claim 2, wherein the essentially
lipophilic agent is selected from the group consisting of n-olein
toluene, xylene, benzene, petroleum, paraffin, carbon disulfide,
chloroform, carbon tetrachloride, trichloro, n-butylacetate,
amylacetate, methyl benzoate, methyl salicylate, cedarwood oil,
clove oil, inhibisol, limonene and terpineol or a mixture
thereof.
12. The process according to claim 2, wherein the dehydration agent
comprises ethanol and isopropanol.
13. The process according to claim 2, wherein the dehydration agent
comprises ethanol and isopropanol and the essentially lipophilic
agent comprises an alkane.
14. The process according to claim 12, wherein the ethanol, the
isopropanol and the essentially lipophilic agent are used in equal
amounts.
15. The process according to claim 1, wherein the impregnation step
(iv) comprises embedding the sample in a medium selected from the
group consisting of agar, gelatine, polyvinylalcohol,
polyetherglycols, polyethylene glycol monostearate, diethylene
glycol distearate, ester wax, polyester wax, nitrocellulose,
paraffin wax or a mixture thereof.
16. The process according to claim 2, wherein the impregnation step
(iv) comprises embedding the sample in a medium selected from the
group consisting of agar, gelatine, polyvinylalcohol,
polyetherglycols, polyethylene glycol monostearate, diethylene
glycol distearate, ester wax, polyester wax, nitrocellulose,
paraffin wax or a mixture thereof.
17. The process according to claim 1, wherein said steps (ii) and
(iii) are carried out under pressure in the range of from 0.11 to
1.0 MPa.
18. The process according to claim 2, wherein said steps (ii) and
(iii) are carried out under pressure in the range of from 0.11 to
1.0 MPa.
19. The process according to claim 1, wherein said steps (ii) and
(iii) are carried out at a temperature in the range of from 70 to
90.degree. C.
20. The process according to claim 2, wherein said steps (ii) and
(iii) are carried out at a temperature in the range of from 70 to
90.degree. C.
21. The process according to claim 17, wherein said steps (ii) and
(iii) are carried out at a temperature in the range of from 70 to
90.degree. C.
22. The process according to claim 18, wherein said steps (ii) and
(iii) are carried out at a temperature in the range of from 70 to
90.degree. C.
23. The process according to claim 4, wherein said step (v) is
carried out at a pressure in the range of from 0.01 to 0.05
MPa.
24. The process according to claim 23, wherein said step (v) is
carried out at a temperature in the range of from 30 to 60.degree.
C.
25. The process according to claim 4, wherein said step (v) is
carried out at a temperature in the range of from 30 to 60.degree.
C.
26. The process according to claim 1, wherein the process is
carried out in a microwave heating device.
27. The process according to claim 2, wherein the process is
carried out in a microwave heating device.
28. The process according to claim 17, wherein the process is
carried out in a microwave heating device.
29. The process according to claim 18 wherein the process is
carried out in a microwave heating device.
30. The process according to claim 19, wherein the process is
carried out in a microwave heating device.
31. The process according to claim 20, wherein the process is
carried out in a microwave heating device.
32. The process according to claim 21, wherein the process is
carried out in a microwave heating device.
33. The process according to claim 22, wherein the process is
carried out in a microwave heating device.
34. The process according to claim 23, wherein the process is
carried out in a microwave heating device.
35. The process according to claim 24, wherein the process is
carried out in a microwave heating device.
36. The process according to claim 25, wherein the process is
carried out in a microwave heating device.
37. The process according to claim 1, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
38. The process according to claim 4, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
39. The process according to claim 26, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
40. The process according to claim 27, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
41. The process according to claim 28, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
42. The process according to claim 29, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
43. The process according to claim 30, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
44. The process according to claim 31, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
45. The process according to claim 32, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
46. The process according to claim 33, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
47. The process according to claim 34, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
48. The process according to claim 35, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
49. The process according to claim 36, wherein said step (iv) is
carried out under alternant pressure and vacuum conditions.
Description
The present invention relates to the processing of organic tissues
to prepare them for further investigation. More specifically the
present invention relates to a process for processing organic
tissue via a combination of microwave, vacuum and pressure
treatment comprising the steps of fixing an organic sample,
dehydrating and clearing the sample, and subsequently infiltrating
and embedding the sample in a supporting medium to prepare it for
basic Haematoxylin and Eosinstans and further techniques such as
microwave staining, immunostaining, decalcification, hybridisation
etc., for subsequent examination by light and electron
microscopy.
Biopsies for diagnostic pathology and for anatomy pathology are
presently prepared according to a conventional method, which is
based on the following essential steps:
1. The tissue to be examined is treated with formaline or a saline
solution to stop the --process of decay and to stabilise the tissue
so as to protect it against physical and chemical rigours of the
processing.
2. In the second step the sample is dehydrated, i.e. some or all of
the free water contained in the specimen ist removed therefrom.
During this procedure various cellular components are dissolved by
the dehydrating fluids, such as lipids, which are extracted, and
water soluble proteins, which are dissolved in aqueous
alcohols.
3. The third step includes the treatment of the tissue with another
solvent, the so-called clearing agent. Clearing is the transition
step between dehydration and infiltration with the supporting
medium. Since many dehydrants are immiscible with said subsequently
applied supporting medium, a solvent is used which is miscible both
with the dehydrant and the embedding medium to facilitate the
transition between dehydration and infiltration steps.
4. The last step in the processing of organic specimens involves
infiltrating the tissue cavities and cells by the supporting
substance and finally embedding the tissue in the medium which will
provide sufficient external support during the ensuing
processing.
These procedures show, however, a variety of disadvantages, such as
a long processing time of the specimens which may amount up to 50
hours per sample. Furthermore, a huge amount of the dehydrating
agent and the clearing agent has to be employed. In a normal
procedure the specimens are treated several times with the
dehydrating agent, e.g. ethanol in rising concentrations (up to
100% ethanol), and subsequently several times with the clearing
agent in order to get rid of the dehydrating agent. Since large
amounts of solvents have to be used, said solvents even being of
high purity, the costs of such procedures are rather high.
A histoprocessing procedure with a shortened processing time is for
example known, from the article "The Two-Step Vacuum-Microwave
Method for Histoprocessing" by M. E. Boon et al., European Journal
of Morphology, Vol. 33, No. 4, 1995, pages 349-358. The
histoprocessing method of organic tissues disclosed therein
includes the steps of fixing the tissue, dehydrating the sample,
clearing the sample and embededing the sample in paraffin, whereby
the dehydrating and the clearing of the sample are carried out
simultaneously in 100%--isopropanol in a vacuum chamber and heated
by microwaves. Isopropanol is used in this step because it is both
a weak dehydrator and a claring agent. In this step the temperature
is controlled at 55.degree. C. and the pressure at 0,04 MPa (400
mbar) so that the tissue can withstand the hazards of the treatment
without adverse affects.
On the other hand it proved to be difficult in practice to properly
process organic tissue samples having a substantial amount of
lipids contained therein (fatty tissue). In this case, the free
water to be removed is retained in the tissue to a large extent
making the use of excessive amounts of dehydrating agent necessary,
which eventually results in an inferior quality of the specimens.
This fact was considered to be due to fat cells embodied in the
respective specimens preventing a free migration of water out of
the tissue.
An alternative technique has been developed to reduce the time
delay between receipt of the biopsy material in the laboratory and
the availability of the tissue for further examination, and to
rapidly obtain thin slides of the respective material. The biopsies
are frozen in liquid nitrogen and then cut at lower temperature in
a cryostat microtome. However, the specimens processed according to
this method show a rather poor quality.
Hence there was a need in the art for an improved process for
processing organic samples which obviates the above problems.
According to an aspect of the present invention there is provided a
process for processing organic tissue which comprises the steps
of
(i) fixing an organic sample to be processed,
(ii) dehydrating the sample with dehydration agent,
(iii) clearing the sample with an essentially lipophilic agent,
and
(iv) impregnating the sample,
wherein said steps (ii) and (iii) are carried out simultaneously by
applying a mixture of the dehydration agent and the essentially
lipophilic agent simultaneously to the sample.
Other aspects and advantages of the present invention will be
readily apparent to the skilled person when reading the present
specification together with the claims taking into account the
accompanying drawings.
The first step in the processing of organic tissue is the fixatikon
of the respective specimen. In the process of the present invention
there is no particular limitation as to the fixation method
utilized. Hence strategies commonly employed to ensure fixation may
be used. Methodes suitable in the process of the present invention
included microwave irradiation of biopsy specimens in normal
saline, continuing fixation on the tissue processor with one or
more changes of the routine fixative, preferably at elevated
temperatures of 40 to 60.degree. C., secondary fixation in formol
sublimate on the tissue processor or in alcoholic fixative which
will complete fixation while initiating dehydration, fixing in
buffered phenol-formaldehyde pH 7.0 and pH 5.5 sequence at
40.degree. C., etc.
In the second step according to the present process the specimens
are treated with a combination of a dehydrating agent and an
essentially lipohilic agent, i.e. a clearing agent.
As the dehydrating agent to be used in the present process there
may be mentioned compounds which are essentially of hydrophilic
nature. Examples for said compounds comprise lower alcohols,
preferably alcohols containing 1 to 8 carbon atoms, lower ethers,
preferably ethers containing 1 to 8 carbon atoms, ketones,
preferably acetone, phenols, anilines, tetrahydrofuran,
2,2-dimethoxypropan, 2,2 diethoxypropan and creosote. Since any of
these compounds reveal specific advantages for a given purpose or
for a specific tissue specimen, the person skilled in the art will
select the appropriate one for the respective task, or a mixture
thereof.
Especially preferred dehydrating agents include alcohols, such as
methanol, ethanol, propanol, isopropanol, n-butanol and t-butanol,
or mixtures of these. Isopropanol or a mixture of isopropanol and
ethanol is most preferred.
As the clearing agent to be used together with the dehydration
agent in the present process there may be mentioned compounds,
which are essentially of amphiphilic or lipophilic nature, which
agents should preferably be miscible with the respective
dehydrating agent utilized. Examples for said clearing agents
include aliphatic hydrocarbons, which may be straight or branched,
and which may be substituted with halides, such as chlorine,
bromine or iodine. Also aromatic hydrocarbons may be used, which
may be substitued by a moiety, comprising from 1 to 4 carbon atoms
or which may be substituted by halides, such as chlorine, bromine
or iodine. Also alkanes, esters and terpenes have been found to be
suitable clearing agents in the present process.
Specific examples include toluene, xylene, benzene, petroleum,
paraffin, carbon disulphide, chloroform, carbon tetrachloride,
trichlorethane, n-butylacetate, amylacetate, methyl benzoate,
methyl salicylate, cedarwood oil, clove oil, inhibisol (inhibisol
is the tradename of a cleaning fluid consisting of
1,1,1-trichlorethane with inhibitors designed to reduce its
toxicity), limonene, terpineol and mixtures thereof. Preferred
agents are aliphatic and aromatic hydrocarbons with said aliphatic
hydrocarbon having from 6 to 12 carbon atoms, preferably 6 to 10,
most preferably n-hexane, n-heptane and n-octane, being more
preferred. Also a preferred clearing agent is n-olein.
Based on his general knowledge and the teaching of the present
invention the person skilled in the art will select the appropriate
combination of the dehydration agent and the clearing agent for a
given purpose. Thus, when treating e.g. fatty tissue a combination
of a dehydration agent and a clearing agent will be chosen which
allows a rapid extraction of lipids.
Preferred combinations of a dehydration agent and a clearing agent
are alcohols combined with alithatic and/or aromatic hydrocarbons.
A combination of alcohols, having 1 to 4 carbon atoms, together
with aliphatic and/or aromatic hydrocarbons is more preferred with
the combination of isopropanol with one or more of petroleum,
ether, n-hexane, n-heptane, n-octane, n-olein, chloroform and/or
acetone being most preferred. Instead of using isopropanol alone a
combination of ethanol and isopropanol is more preferably
employed.
The amount of the dehydrating agent and the clearing agent in the
solvent combination utilized may vary depending on the tissue to be
treated and the properties inherent to the respective agents. A
ratio of dehydrating agent: clearing agent of 20-80:80-20% by
volume are within the scope of the present invention. A preferred
ration is 50:50 more preferred 60:40. In case of the preferred
mixture of ethanol and isopropanol and an alkane all constituents
are preferably included in equal amounts by volume. Unexpectedly it
has been shown that the use of isopropanol in the
dehydration/clearing agent results in the sample to be easily cut
for later investigation.
The dehydration/clearing step is preferably carried out at a
pressure of from atmospheric pressure to 1 MPa (10 bar), preferably
at slightly elevated pressure, i.e. 0.11 to 0.5 MPa, and up to 1
MPa. Treatment under pressure allows for the sample to be treated
at higher temperatures, namely at a temperature of from room
temperature up to 90.degree. C., preferably 70 to 90.degree. C.,
more preferably 70 to 85.degree. C., most preferred at a
temperature of from 80 to 85.degree. C. without causing the
combination of dehydration/clearing agent to evaporate and
disrupting the tissue structure. The pressure may be effected by
simply heating the sample in a closed container with the
evaporating liquids building up a slight pressure. It is, however,
preferred to build up the pressure by introducing a gas in the
container, such as an inert gas, preferably nitrogen gas or carbon
dioxide.
Surprisingly it has been found that the above conditions of
pressure and temperature also result in a smoother defatting of
samples without causing a denaturation of proteins and cell
structures in the sample. Thus the morphology of the cell is fully
maintained. Further this step is preferably carried out in a
microwave device since it has been shown that heating of the sample
may smoothly be effected by microwave treatment.
Before carrying out the impregnation step the sample is dried, said
process being primarily effected by means of moderate heat, i.e.
heating the sample at a temperature from 30 to 60.degree. C.,
preferably 60.degree. C. In order to promote the removal of
residula water and solvent the drying process should be carried out
under reduced pressure, preferably at a pressure in the range of
from 0.01 to 0.05 MPa, more preferably at 0.01 MPa. Methodes
currently employed also involve a quick rinse of the moist tissue
cassettes with alcohol, preferably ethanol, no less than 80%
concentration.
In the impregnation step (iv) of the present process the sample is
infiltrated by and embedded in a medium the properties of which
should generally approach those of the tissues to be examined with
regard to density, elasticity, plasticity, viscosity and adhesion
and should be harmless to the embedded material. Generally the
infiltration medium and the embedding medium are equal, but they
may be different as well. Suitable materials to be used as
infiltration and/or as embedding medium include compounds selected
from the group consisting of agar, gelatine, polyvinylalcohol,
polyetherglycols, polyethylene glycol monostearate, diethylene
glycol distearate, ester wax, polyester wax, nitrocellulose,
paraffin wax or a mixture thereof. Additives may be added to the
waxes or blends of waxes, such additives being e.g. Piccolyte 115,
which is a thermoplastic terpene resin added at the rate of 5-10%
to the infiltrating wax, plastic polymers, such as polyethylene wax
or dimethylsuloxide (DMSO).
When applying the supporting media to the sample, vacuum should
preferably be applied to remove air entrapped in the tissue and to
remove residual solvents used in the preceding combined dehydration
and clearing step. Thus the impregnation step is preferably
conducted under vacuum, starting with a moderate vacuum of from
about 0.05 to 0.02 MPa, preferably 0.03 to 0.02 MPa, in order to
carefully degas the molten paraffin applied to the sample. In a
next step the vacuum is then lowered, preferably to about 0.01 MPa
or even 0.005 MPa.
Alternatively the impregnation step may also be carried out by
alternately applying a moderate pressure, such as from 0.1 to 0.5
MPa, preferably 0.11 to 0.3 MPa for a time period of about 5
minutes or less, preferably 3 or 4 minutes, more preferred about 2
minutes, and a moderate vacuum for about the same time period. The
reduced pressure to be applied ranges of from 0.01 to 0.08 MPa,
preferably from 0.01 to 0.05 MPa, more preferred from 0.02 to 0.03
MPa.
According to the present process it became possible to process a
variety of tissues including difficult tissues, such as tissues
which are largely composed of keratin, dense collagen, closely
packed smooth muscle fibres, colloid areas of haemorrhage, thrombi
or yolk (hard dense tissues), or mummified tissues or yolk rich
tissues and also fatty tissues. In addition it could be shown that
the processing time for the specimens could be reduced enormously
to about 30 minutes for processing needle biopsies and about 60 to
120 minutes for standard type tissue specimens.
Surprisingly it has been shown that the properties of the
respective dehydrating agents and clearing agents are not altered
when being combined, thus allowing a simple and efficient
dehydration and clearing at the same time. In case of fatty tissues
it has furthermore been shown that dehydration may easily be
accomplished since the clearing agent also serves for extracting
lipid substances, resulting in a smooth removal of the water
without deterioration of the structure of the tissue to be
treated.
In a preferred embodiment the present process is carried out in a
microwave heating device. Microwave treatment has been shown to
accelerate the action of cross-linking fixatives and also allows
the removal of the dehydrating agent and the clearing agent to
proceed more smoothly.
According to another aspect of the present invention there is
provided an apparatus for carrying out the above process which
comprises
(i) a microwave heating device comprising a sealable processing
chamber arranged therein,
(ii) a container arranged in said processing chamber for loading
the samples,
(iii) a means for applying vacuum to said processing chamber,
(iv) a means for applying pressure to said processing chamber,
and
(v) a line for applying vacuum or pressure to said processing
chamber,
wherein the processing chamber comprises a plate and an
automatically liftable dome, wherein said liftable dome encloses
the container arranged in said processing chamber, and wherein the
line for applying vacuum or pressure to said processing chamber
extends through said dome.
In a preferred embodiment one single vacuum/pressure pump unit may
be used for applying pressure and vacuum to the processing chamber
and for the up and down movement of the dome. Further, the
processing chamber may be rotatable by 360.degree. for ensuring a
uniform microwave radiation of the samples in the microwave heating
device.
Other means for executing said apparatus and preferred embodiments
thereof are illustrated in the copending PCT application
WO95/19560, the contents of which is fully incorporated herein by
way of reference.
With reference to the drawings, the present invention will be
further illustrated in the following by way of example, wherein
FIG. 1 is a schematic drawing showing the dehydrating and clearing
step of a sample;
FIG. 2 is a schematic drawing showing the drying step of a sample;
and
FIG. 3 is a schematic drawing showing the impregnating step of a
sample.
The histoprocessing procedure is preferably carried out in a
microwave heating device with a microwave generator including a
magnetron as a main component thereof for generating microwaves
which are by means of a wave guide coupled into the interior of the
microwave heating device.
The microwave heating device comprises an access opening and
incorporates a rotating glass processing chamber 1, 2 which
accomodates up to sixty tissue cassettes contained in a
microwave-transparent rack preferably made of PTFE
(polytetrafluorethylene), arranged as two concentric circles inside
a glass container 3.
The processing chamber 1, 2 consists of a rotatable carousel 2 on
the bottom of the microwave heating device and a dome 1 which can
be raised and lowered pneumatically so that the glass container 3
can be introduced through the access opening in the microwave
heating device into the processing chamber. The processing chamber
1, 2 is connected via connecting means with a vacuum/pressure pump
unit, and it comprises a sealing arrangement to close the
processing chamber in a vacuum-tight or pressure-tight manner,
respectively. Preferably, the vacuum/pressure pump unit causes also
the raising and lowering of the dome 1.
The microwave heating device further comprises a temperature
sensor, e.g. an IR temperature sensor combined with an IR window in
the sidewall of the glass dome, for monitoring the temperature of
the chemical solution within the container 3. Alternatively, a
fiber optic sensor may be used which is located in the central
column of the dome 1 and which is connected to the dome 1 to be
raised and lowered together with the dome.
The gas evacuated from the processing chamber 1, 2 is preferably
passed through a charcoal filter. An automatic solvent detector may
be included in the present apparatus to signal the exhaustion of
the charcoal.
The microwave heating device as well as the vacuum/pressure pump
unit and the temperature sensor are connected by means of
connecting means with and controlled through an interactive touch
screen computer and dedicated software which ensures consistency
and simplicity of operation. A menu of processing schedules can be
activated by touch screen icons with preset parameters for
microwave power, time, temperature, vacuum and pressure, all
continously monitored and regulated by the computer software. The
histoprocessing procedure is simply started by activating the
relevant icon on the touch screen computer, and the completion of a
selected procedure is indicated by an audible alarm. The system
also provides protection of stored programs witch access by means
of a key card, ensuring only authorized access of modification or
creation of customized processing schedules.
In the following, the histoprocessing procedure of tissues will be
exemplarily explained.
Multiple tissue blocks were prepared from a wide range of
formalin-fixed surgical specimens, representing malignant and
benign tissues, including breast, stomach, uterus and spleen. Three
size formats were prepared, representing tissue blocks routinely
encountered in surgical pathology laboratories:
1. Needle and endoscopic biopsies--measuring up to 20 mm in length
and up to 2.5 mm in diameter
2. Intermediate size--measuring up to 15.times.10.times.2.5 mm
3. Large size--measuring up to 25.times.20.times.4 mm
Up to sixty tissue sections in universal plastic cassettes may be
placed in a microwave-transparent plastic rack preferably on the
basis of PTFE, followed by a rinse in absolute ethanol to remove
coating formalin and water. Next, the rack is placed into the
circular glass dish 3 containing a solvent mixture of absolute
ethanol, isopropanol and an alkane in a ratio of 1:1:1, a so-called
"JFC solution" which is a proprietary name of Milestone/MLS
Sorisole, Italy. A PTFE based cover (not shown) is placed on the
container 3.
Then the dome 1 of the processing chamber is lifted pneumatically
and the container 3 is introduced in the microwave heating device
under the glass dome, with the appropriate processing schedule
selected. The processing schedule chosen was determined by the
largest piece of tissue in the batch being processed. The entire
process consisted of three main steps with each controlled by its
own preset program, as shown in the FIGS. 1 to 3.
In the first step shown in FIG. 1, i.e. the dehydration and
clearing of the tissues, the JFC solution extracts lipids from
fatty tissues releasing the cells, so that under the influence of
the microwave the temperature of the water in the cells increases,
thereby reducing its viscosity and resulting in a migration of the
water towards the external periphery of the cell from where it
moves into the solvent solution. This happens in a temperature
range which is physiological and not damaging to the tissues.
This step is carried out under pressure of approximately 0.2 MPa to
eliminate vaporization of reagents with low boiling point, keeping
the concentration constant. As for this reason the temperature can
be higher than with known histoprocessing procedures, i.e. up to
80.degree. C.
The dehydration and clearing time of step 1 was for the needle and
endoscopic biopsies up to 18 minutes, for intermediate size up to
65 minutes and for large size up to 90 minutes.
At the end of the above first step the dome 1 is automatically
raised, the container 3 is taken out from the processing chamber
and the microwave heating device and the rack with the cassettes is
placed into an empty glass dish 3, this time without a PTFE cover.
The container 3 is repositioned under the glass dome 1 in the
microwave heating device and a drying program is started by the
computer.
In the second step of drying the tissues shown in FIG. 2, the
tissues are prepared for the final wax impregnation, i.e. the
remaining JFC solution in the tissues must be removed. This step is
carried out under high vacuum of approximately 0.01 MPa and a
temperature of about 60.degree. C. The vacuum lowers the boiling
and vaporization point temperatures of the JFC reagent and produces
complete tissue dehydration and the evaporation of the JFC
reagent.
The drying time of step 2 was for the needle and endoscopic
biopsies up to 3 minutes and for intermediate and large size up to
5 minutes.
At the end of the above drying step the dome 1 is lifted
automatically and the container 3 is taken out from the processing
chamber and the microwave heating device. The rack with the plastic
cassettes is transferred to a preheated container 3 containing
histology grade molten wax of about 65.degree. C. as well as
heating elements of WEFLON at the bottom of the container 3 itself.
The addition of the heating elements is required to avoid
solidification of paraffin which does not absorb microwaves. A
WEFLON cover (not shown) is placed on the glass container 3 which
is again placed in the microwave heating device under the glass
dome 1.
In the third step of wax impregnation shown in FIG. 3 any residual
traces of JFC solution within the tissues are removed by
substitution with molten wax under vacuum of approximately 0.01
MPa. The temperature is set at about 65.degree. C.
The impregnation time of step 3 was for the needle and endoscopic
biopsies up to 9 minutes, for intermediate size up to 20 minutes
and for large size up to 25 minutes.
On completion of step 3 the dome 1 is lifted automatically and the
container 3 is taken out from the processing chamber and the
microwave heating device. The tissues within the cassettes are now
ready for the conventional embedding, cutting and staining
procedures to obtain slides.
The total processing time to paraffin for the specimens were:
needle and endoscopic biopsies 30 minutes, intermediate size 90
minutes and large size 120 minutes.
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